Development of one-step multiplex qPCR/RT-qPCR assays for simultaneous detection of SARS-CoV-2 and other viral and bacterial agents associated with canine and feline respiratory disease
THIEULENT C. 1,2, CAROSSINO M. 1,2, PEAK L. 2, STROTHER K. 2, LI G. 3, WOLFSON W. 4, BALASURIYA U. 1,2
1 Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, United States; 2 Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, United States; 3 Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, United States; 4 Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, United States
Canine infectious respiratory disease complex (CIRDC) and feline upper respiratory tract disease (URTD) are the primary causes of respiratory disease in companion animals and are associated with a wide array of viruses and bacteria acting as either individual etiologic agents or in combination, making etiologic diagnosis challenging. Additionally, SARS-CoV-2 has been reported to infect both dogs and cats. Therefore, the rapid detection and differentiation of SARS-CoV-2 from other common viral and bacterial agents in a single specimen is critical. Methods
Here, two panels of one-step TaqMan® multiplex qPCR/RT-qPCR were developed and validated for the identification of CIRDC and feline URTD-associated agents along with SARS-CoV-2. The canine respiratory panel included the detection of 8 viruses and 4 bacteria. The feline respiratory panel included the detection of 4 viruses and 3 bacteria. The analytical performance of each assay was evaluated using reference strains of each pathogen and plasmid DNA or in vitro transcribed RNA containing the target sequences. The clinical performance was evaluated using 76 and 63 nasal/nasopharyngeal swabs obtained from CIRDC-suspected dogs and URTD-suspected cats, respectively. Results
All the multiplex assays demonstrated high specificity, analytical sensitivity, efficiency, and linearity. Among the clinical samples derived from dogs, Mycoplasma canis, M. cynos and canine respiratory coronavirus were the most prevalent pathogens. The emerging canine pneumovirus was detected in four samples. Among the clinical samples from cats, M. felis was the most prevalent pathogen, followed by feline herpesvirus type-1, Chlamydia felis and feline calicivirus. SARS-CoV-2 was detected in four canine and two feline samples. A high prevalence of co-infection was detected, with a rate of 29% and 59% in the canine and feline respiratory samples, respectively. Conclusions
These two newly panels of one-step TaqMan® multiplex qPCR/RT-qPCR are useful and reliable for the rapid detection and identification of canine and feline respiratory pathogens, along with SARS-CoV-2. The high prevalence of co-infection highlights the need for the simultaneous detection of multiple pathogens using such panels in order to implement proper treatment and/or prevention plans.